Accomplishment 1. Elucidate Mechanisms Recovering Stressed Mitochondria by Regulating ER-mitochondrial Contacts (Puri et al., Nature Communications 2019) Chronic mitochondrial stress associates with major neurodegenerative diseases. Recovering stressed mitochondria constitutes a critical step of mitochondrial quality control and thus energy maintenance in early stages of neurodegeneration. Parkin-mediated mitophagy is a key cellular pathway to eliminate damaged mitochondria in many non-neuronal cells. Our previous studies revealed that Parkin-mediated mitophagy is observed only in a small portion of mature neurons and occurs much more slowly than in non-neuronal cells (Cai et al., Current Biology 2012; Lin et al., Neuron 2017). These findings argue for unique mechanisms that maintain and recover neuronal mitochondrial integrity and thus energy homeostasis, rather than activate mitophagy. In order to support this assumption, we addressed three fundamental questions: (1) Can neurons recover chronically stressed mitochondria before Parkin-mediated mitophagy is activated? (2) Is mitophagy the last resort for mitochondrial quality control after recovery mechanisms have failed? (3) If this is the case, does mitochondrial ubiquitin ligase 1 (Mul1) play an early role in maintaining neuronal mitochondrial integrity? Addressing these questions is relevant to several major neurodegenerative diseases that associate with chronic mitochondrial stress. We reveal that Mul1-Mfn2 pathway maintains neuronal mitochondrial integrity through its dual-role in regulating mitochondrial morphology and ER-Mitochondria contacts. This mechanism ensures that mitophagy degradation is restrained in neurons under early stress conditions. Mul1 deficiency increases Mfn2 activity that triggers the first phasic mitochondrial hyperfusion and also acts as an ER-Mitochondria tethering antagonist. Reduced ER-Mitochondria coupling leads to increased cytoplasmic Ca2+ load that activates calcineurin and induces the second phasic Drp1-dependent mitochondrial fragmentation and mitophagy. Overexpressing Mfn2, but not Mfn1, mimics Mul1-deficient phenotypes, while expressing PTPIP51, an ER-Mitochondria anchoring protein, suppresses Parkin-mediated mitophagy. Thus, by regulating mitochondrial morphology and ER-Mitochondria contacts, Mul1-Mfn2 pathway plays an early checkpoint role in maintaining mitochondrial integrity and restrains parkin-mediated mitophagy in mature neurons. Our study provides new mechanistic insights into neuronal mitochondrial maintenance under stress conditions. Identifying this pathway is particularly relevant because chronic mitochondrial dysfunction and altered ER-Mitochondria contacts have been reported in major neurodegenerative diseases including Alzheimers and Parkinsons diseases, amyotrophic lateral sclerosis, and hereditary spastic paraplegia. Accomplishment 2. Elucidate Mechanisms Promoting CNS Regeneration and Functional Recovery after Spinal Cord Injuries (Han et al., manuscript was submitted and under invited revision) Mature CNS axons in adult mammals typically fail to regenerate after spinal cord and brain injuries, leading to permanent neurological impairments. The underlying mechanisms that account for declined regenerative capacity following CNS injuries remain largely unknown. For successful regeneration to occur, injured axons undergo multiple intracellular repair processes, including the resealing of injured terminals, reconstruction of cytoskeleton, synthesis and transport of building materials, assembly of axonal components, and formation of growth cones and synapses. All of these events require high levels of energy consumption. Our previous work revealed SNPH as a static anchor protein, holding axonal mitochondria stationary via microtubule interactions (Kang et al., Cell 2008). SNPH expression is increased in mature neurons, thus contributing to declined transport of axonal mitochondria in adult CNS in vivo (Zhou et al., 2016). Deleting snph in mouse robustly increases axonal mitochondrial motility in vitro and in vivo. While anchored mitochondria in distal axons serve as local energy sources, axonal injury is a strong stressor that locally induces acute mitochondrial damage. Insufficient energy metabolism and ATP supply when mitochondria are damaged and increased energy consumption during regeneration lead to energy deficits in injured axons. Our hypothesis is that elevation of SNPH expression in mature CNS neurons and reduced axonal mitochondrial transport accelerate local crisis of energy metabolism in injured axons, thus accounting for declined regenerative capacity in mature CNS neurons. Such a local energy supply may be particularly critical to support regenerative growth when injury occurs to distal long-projection axons. Thus, enhancing mitochondrial transport would help remove damaged mitochondria and replenish healthy ones in injured axons to meet increased energy requirements during regeneration. To test our hypothesis directly in spinal cord injury (SCI) models, we collaborated with Dr. Xiao-Ming Xus lab at Department of Neurological Surgery, Indiana University, a leading expert in SCI research. We had the unique experimental advantage of utilizing three different in vivo SCI models to study axonal regeneration in the genetic snph-/- mice, in which axonal mitochondrial transport is robustly increased in vivo. We asked whether recovering local mitochondrial integrity and reversing energy metabolism facilitate in vivo CNS axonal regeneration following injury. Using three different CNS injury mouse models, we demonstrate that snph-/- mice display enhanced corticospinal tract (CST) regeneration passing through a spinal cord lesion, accelerated regrowth of monoaminergic axons across a complete spinal transection gap, and increased compensatory sprouting of uninjured CST. Notably, regenerating CST axons are able to form functional synapses, transmit electrophysiological signals, and promote motor functional recovery. Our energy crisis model is further supported by the finding that systemic administration of creatine, a bioenergetic compound, significantly facilitates CST regeneration. Thus, our study provides a new mechanistic insight into intrinsic regeneration failure in the CNS and suggests that enhancing local energy recovery is a promising strategy to promote axonal regeneration and functional recovery after CNS injuries. Our study established, for the first time, that SCI-induced mitochondrial dysfunction and associated crisis of energy metabolism is an intrinsic mechanism linked to declined regenerative capacity in the adult mammalian CNS. Enhanced local ATP supply is critical to meeting the metabolic requirements of axon regeneration. Enhanced mitochondrial transport not only helps to remove injured mitochondria, but also replenishes healthy ones to distal injured axons, thus recovering mitochondrial integrity and rescuing energy deficits. Thus, activating intrinsic regrowth programs requires recovery of energy deficits by enhancing mitochondrial transport or by elevating local energy metabolism. Targeting energy metabolism may represent a new and promising therapeutic direction to stimulate axon regeneration and functional recovery after CNS injuries, such as those in the spinal cord.